Topoisomerase II

In HEK 293 cells and Xenopus oocytes, amsacrine (m-AMSA) inhibits HERG currents in a concentration-dependent manner with IC50 values of 209.4 nm and 2.0 μM, respectively. The voltage dependency of activation (-7.6 mV) and inactivation (-7.6 mV) is shifted negatively by amsacrine (m-AMSA). Amsacridine's HERG current blockage is frequency independent [1]. Increased chromosomal abnormalities, ranging from 8% to 100%, and increased SCE, which was normal at the lowest concentration examined 1.5 times the value, were seen in vitro tests utilizing varying concentrations of m-AMSA on normal human cells. 12 times the typical value (0.25 μg/mL) [3], or 0.005 μg/mL. The induction of amacridine (m-AMSA)-induced apoptosis in U937 cells is distinguished by the activation of caspase-9 and caspase-3, elevation of intracellular Ca2+ concentration, depolarization of the mitochondria, and downregulation of MCL1. By decreasing MCL1 stability, amsacrine (m-AMSA) causes MCL1 downregulation. Moreover, U937 cells treated with amsacridine exhibited Ca2+-mediated ERK inactivation and AKT degradation [4].

The frequency of micronucleated polychromatic erythrocytes rose significantly after treatment with 9 and 12 mg/kg of amsacrine in rats treated with varying doses (0.5-12 mg/kg). Furthermore, this study shows that nocodazole has a high incidence of clastogenicity and a low incidence during the mitotic phase in vivo, whereas m-AMSA has a high incidence and a low incidence. incidence [2].

1 The topoisomerase II inhibitor amsacrine is used in the treatment of acute myelogenous leukemia. Although most anticancer drugs are believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by reports of QT interval prolongation, ventricular fibrillation and death associated with amsacrine treatment. Since blockade of cardiac human ether-a-go-go-related gene (HERG) potassium currents is an important cause of acquired LQTS, we investigated the acute effects of amsacrine on cloned HERG channels to determine the electrophysiological basis for its proarrhythmic potential. 2 HERG channels were heterologously expressed in human HEK 293 cells and Xenopus laevis oocytes, and the respective potassium currents were recorded using patch-clamp and two-microelectrode voltage-clamp electrophysiology. 3 Amsacrine blocked HERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner, with IC50 values of 209.4 nm and 2.0 microm, respectively. 4 HERG channels were primarily blocked in the open and inactivated states, and no additional voltage dependence was observed. Amsacrine caused a negative shift in the voltage dependence of both activation (-7.6 mV) and inactivation (-7.6 mV). HERG current block by amsacrine was not frequency dependent. 5 The S6 domain mutations Y652A and F656A attenuated (Y652A) or abolished (F656A, Y652A/F656A) HERG current blockade, indicating that amsacrine binding requires a common drug receptor within the pore-S6 region. 6 In conclusion, these data demonstrate that the anticancer drug amsacrine is an antagonist of cloned HERG potassium channels, providing a molecular mechanism for the previously reported QTc interval prolongation during clinical administration of amsacrine.[1]

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Previous studies have attributed the anticancer activity of amsacrine to its inhibitory effect on topoisomerase II. However, 9-aminoacridine derivatives, which have the same structural scaffold as amsacrine, induce cancer cell apoptosis by altering the expression of BCL2 family proteins. Therefore, in the present study, we assessed whether BCL2 family proteins mediated the cytotoxic effects of amsacrine on human leukemia U937 cells. Amsacrine-induced apoptosis of U937 cells was characterized by caspase-9 and caspase-3 activation, increased intracellular Ca2+ concentration, mitochondrial depolarization, and MCL1 down-regulation. Amsacrine induced MCL1 down-regulation by decreasing its stability. Further, amsacrine-treated U937 cells showed AKT degradation and Ca2+-mediated ERK inactivation. Blockade of ERK-mediated phosphorylation of MCL1 inhibited the effect of Pin1 on the stabilization of MCL1, and AKT degradation promoted GSK3β-mediated degradation of MCL1. Restoration of ERK phosphorylation and AKT expression abrogated amsacrine-induced MCL1 down-regulation. Moreover, MCL1 over-expression inhibited amsacrine-induced depolarization of mitochondria membrane and increased the viability of amsacrine-treated cells. Taken together, our data indicate that amsacrine abolishes ERK- and Pin1-mediated stabilization of MCL1 and promotes GSK3β-mediated degradation of MCL1, leading to activate mitochondria-mediated apoptosis pathway in U937 cells[4].

The mechanism of genotoxic potential of the cancer chemotherapeutic drugs amsacrine and nocodazole in mouse bone marrow was investigated using a micronucleus test complemented by fluorescence in situ hybridization assay with mouse centromeric and telomeric DNA probes. In animals treated with different doses of amsacrine (0.5-12 mg kg(-1) ), the frequencies of micronucleated polychromatic erythrocytes increased significantly after treatment with 9 and 12 mg kg(-1) . A statistically significant increase in micronuclei frequency was also detected for 75 mg kg(-1) nocodazole (two exposures, spaced 24 h apart). Both compounds caused significant suppressions of erythroblast proliferation at higher doses. Furthermore, the present study demonstrated for the first time that amsacrine has high incidences of clastogenicity and low incidences of aneugenicity whereas nocodazole has high incidences of aneugenicity and low incidences of clastogenicity during mitotic phases in vivo. The assay also showed that chromosomes can be enclosed in the micronuclei before and after centromere separation. Therefore, the clinical use of these genotoxic drugs must be weighed against the risks of the development of chromosomal aberrations in cancer patients and medical personnel exposed to drug regimens that include these chemicals.[2]

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Amsacrine (m-AMSA) is presently being utilized in phase I-II studies at the Medicine Branch, National Cancer Institute, National Institutes of Health (Bethesda, MD), and is being administered as a continuous infusion to patients with progressive malignancy after conventional therapy. In the present study, we examined the effects of this drug, in vivo and in vitro, on chromosomal morphology and the frequency of sister chromatid exchange (SCE) induction in human peripheral blood lymphocytes. In the in vivo studies, eight patients receiving 30 mg/m2/day of m-AMSA by continuous infusion showed increased levels of chromosomal aberrations, up to a maximum of 14% (median; range, 10%-24%) at 96 hours compared to 1% (median; range, 0%-4%) in the control group; no increase was noted in SCE frequencies.[3]

Absorption, Distribution and Excretion
Poorly absorbed
Volume of distribution (VolD) -- 1.67 L/kg. Amsacrine does not significantly penetrate into the CNS
Elimination: Renal: 35% of the dose is excreted by the kidneys within 72 hours after administration (20% as intact drug). Biliary: Amsacrine is also eliminated by biliary excretion.
In cancer patients, amsacrine undergoes biphasic elimination, with a distribution half-life of 0.25-1.6 hours and an elimination half-time of 4.7-9 hours. The total plasma clearance rate is 200-300 ml/min per sq m, and the apparent distribution volume is 70-110 l/sq m, suggesting concentration in tissues. During a 1 hour injusion of amasacrine at 90-200 mg/sq m, the peak plasma concentration was 10-15 umol/l.
Although not fully reported, early trials in which amsacrine was given orally failed to reach the maximum tolerated dose, as shown by lack of toxicity even at doses as high as 500 mg/sq m per day, suggesting incomplete or erratic absorption. In subsequent studies, the intravenous route was used, with which the maximum tolerated dose in patients with solid tumors is 100-150 mg/sq m when administered over 1-3 hours.
After intravenous administration of (14)C amsacrine to mice and rats, > 50% of the radiolabel was excreted in bile within the first 2 hours, and the bile:plasma ratio was > 400:1; 74% of an intravenous dose was excreted in the feces of mice with 72 hours. These studies demonstrate the importance of the liver in clearance of amsacrine.

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Metabolism / Metabolites
Extensive, primarily hepatic, converted to glutathione conjugate.
Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.
In mouse bile, 5'- and 6'-glutathione conjugates were present in roughly equal amounts and accounted for 70% of the excreted biliary radiolabel after administration of radiolabelled amsacrine. In rats, the principal biliary metabolite was the 5'-gutathione conjugate, which accounted for 80% of the excreted radiolabel within the first 90 minutes and > 50% of the administered dose over 3 hours. The 6'-conjugate was also subsequently identified in rat bile. In rat liver microsomes and human neutrophils, intermediate oxidation products have been identified as N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexadience-1',4'-dii mine and 3'-methoxy-4'-(9-acridinylamino-2'5'-cyclohexadien-1'-one.
Extensive, primarily hepatic, converted to glutathione conjugate.
Half Life: 8-9 hours

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Biological Half-Life
8-9 hours

Toxicity Summary
Amsacrine binds to DNA through intercalation and external binding. It has a base specificity for A-T pairs. Rapidly dividing cells are two to four times more sensitive to amsacrine than are resting cells. Amsacrine appears to cleave DNA by inducing double stranded breaks. Amsacrine also targets and inhibits topoisomerase II. Cytotoxicity is greatest during the S phase of the cell cycle when topoisomerase levels are at a maximum.

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Toxicity Summary
Amsacrine binds to DNA through intercalation and external binding. It has a base specificity for A-T pairs. Rapidly dividing cells are two to four times more sensitive to amsacrine than are resting cells. Amsacrine appears to cleave DNA by inducing double stranded breaks. Amsacrine also targets and inhibits topoisomerase II. Cytotoxicity is greatest during the S phase of the cell cycle when topoisomerase levels are at a maximum.

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Evidence for Carcinogenicity
Evaluation: There is inadequate evidence in humans for the carcinogenicity of amsacrine. There is sufficient evidence in experimental animals for the carcinogenicity of amsacrine. Overall evaluation: Amsacrine is possibly carcinogenic to humans (Group 2B).

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mouse LD50 oral 181 mg/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986
mouse LD50 intraperitoneal 20560 ug/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986
mouse LD50 subcutaneous 110 mg/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986

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Protein Binding
96-98%

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Toxicity Data
Human(iv): TD L0: 12 mg/kg
Mouse(po): LD50: 53420 ug/kg
Mouse(ip): LD50: 15470 µg/kg
Mouse(sc): LD50: 110 mg/kg
Mouse(iv): LD50: 33700 µg/kg
Dog(po): LD50: 50 mg/kg
Dog(iv): LD50: 6250 ug/kg

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Interactions
Leukopenic and/or thrombocytopenic effects of amsacrine may be increased with concurrent or recent therapy if these medications /blood dyscrasia-causing medications/ cause the same effects; dosage adjustment of amsacrine, if necessary, should be based on blood counts.
Additive bone marrow depression, including severe dermatitis and/or mucositis, may occur; dosage reduction may be required when two or more bone marrow depressants, including radiation, are used concurrently or consecutively.

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Non-Human Toxicity Values
LD50 Mouse intraveneous 33.7 mg/kg
LD50 Dog oral 50 mg/kg
LD50 Mouse subcutaneous 110 mg/kg
LD50 Mouse intraperitoneal 15,470 ug/kg
LD50 Mouse oral 53,420 ug/kg

[1]. Inhibition of cardiac HERG currents by the DNA topoisomerase II inhibitor amsacrine: mode of action. Br J Pharmacol. 2004 Jun;142(3):485-94.

[2]. Attia SM. Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine and nocodazole in mouse bone marrow cells. J Appl Toxicol. 2013 Jun;33(6):426-33.

[3]. Cytogenetic effects of amsacrine on human lymphocytes in vivo and in vitro. Cancer Treat Rep. 1984 Jul-Aug;68(7-8):989-97.

[4]. Amsacrine-induced apoptosis of human leukemia U937 cells is mediated by the inhibition of AKT- and ERK-induced stabilization of MCL1. Apoptosis. 2016 Oct 19.

Therapeutic Uses
Cytostatic agent with antiviral and immunosuppressive properties.
Amsacrine is indicated for induction of remission in acute adult leukemia refractory to conventional therapy. /Included in US product labeling/
118 patients with acute leukemias, including initial, relapsed and refractory cases, were treated with domestic Amsacrine (m-AMSA), singly or combined with other drugs. The total CR rate was 39.5% in ALL and 38.8% in ANLL, the response rate was 47.5% for both types of acute leukemias. The CR rate of relapsed and refractory ALL and ANLL treated with combination chemotherapy including domestic m-AMSA was 30.8% and 46.2% respectively. Domestic m-AMSA was similar to the foreign product and many other antitumor drugs in side effects and toxicity. The pharmacokinetics parameters of the drugs, C12h/C6h,K21 and Cmax were correlated with the therapeutic effectiveness
The synthetic aminoacridine derivative amsacrine (m-AMSA) is capable of preventing DNA from serving as a template in replication and DNA synthesis. This mechanism of action is similar to that of anthracyclines, but clinical evidence suggests the lack of cross-resistance. The recommended dosage in patients with solid tumors is 90-120 mg/sq m intravenously every 3-4 weeks. Despite the initial encouraging reports from experimental models, m-AMSA has shown no real impact in the treatment of patients with a wide variety of solid tumors. In relapsed acute nonlymphocytic leukemia, 20-30% of patients will achieve complete remission. An increased remission rate is obtained when m-AMSA is combined with other agents, especially with high-dose cytosine arabinoside, with a complete remission rate of 50-60% in relapsed patients. Currently, several phase III trials are evaluating m-AMSA combinations against daunorubicin-containing regimens in patients with previously untreated acute leukemia. The potential role of these regimens in this disease remains to be defined.
For more Therapeutic Uses (Complete) data for AMSACRINE (7 total), please visit the HSDB record page.

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Drug Warnings
Human systemic effects by intravenous route: nausea or vomiting, thrombosis distant from the injection site, and bone marrow changes.
Although very little information is available regarding distribution of antineoplastic agents into breast milk, breast-feeding is not recommended during chemotherapy because of the potential risks to the infant (adverse effects, mutagenicity, carcinogenicity).
No information is available on the relationship of age to the effects of amsacrine in geriatric patients. However, elderly patients are more likely to have age-related renal function impairment, which may require adjustment of dosage in patients receiving amsacrine.
The bone marrow depressant effects of amsacrine may result in an increased incidence of microbial infection, delayed healing, and gingival bleeding. Dental work, whenever possible, should be completed prior to initiation of therapy or deferred until blood counts have returned to normal. Patients should be instructed in proper oral hygiene, including caution in use of regular toothbrushes, dental floss, and toothpicks.
For more Drug Warnings (Complete) data for AMSACRINE (19 total), please visit the HSDB record page.

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Pharmacodynamics
Amsacrine is an aminoacridine derivative that is a potent intercalating antineoplastic agent. It is effective in the treatment of acute leukemias and malignant lymphomas, but has poor activity in the treatment of solid tumors. It is frequently used in combination with other antineoplastic agents in chemotherapy protocols. It produces consistent but acceptable myelosuppression and cardiotoxic effects.

C21H19N3O3S

393.4589

393.114

C, 64.11; H, 4.87; N, 10.68; O, 12.20; S, 8.15

51264-14-3

Amsacrine hydrochloride;54301-15-4; 54301-16-5 (mesylate) 80277-11-8 (lactate); 80277-07-2 (gluconate); 51264-14-3

2179

Orange to red solid powder

1.4±0.1 g/cm3

563.0±60.0 °C at 760 mmHg

230-240ºC

294.3±32.9 °C

0.0±1.5 mmHg at 25°C

1.723

2.12

2

6

5

28

601

0

XCPGHVQEEXUHNC-UHFFFAOYSA-N

InChI=1S/C21H19N3O3S/c1-27-20-13-14(24-28(2,25)26)11-12-19(20)23-21-15-7-3-5-9-17(15)22-18-10-6-4-8-16(18)21/h3-13,24H,1-2H3,(H,22,23)

N-[4-(acridin-9-ylamino)-3-methoxyphenyl]methanesulfonamide

amsacrine; 51264-14-3; Amsidine; m-AMSA; Amsidyl; Acridinylanisidide; Lamasine; Amekrin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~9.3 mg/mL (~23.64 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)